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SCIENCE CHINA Life Sciences, Volume 61, Issue 9: 1030-1038(2018) https://doi.org/10.1007/s11427-017-9290-1

Loss of Hox5 function results in myofibroblast mislocalization and distal lung matrix defects during postnatal development

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  • ReceivedFeb 20, 2018
  • AcceptedMar 23, 2018
  • PublishedApr 27, 2018

Abstract

Alveologenesis is the final stage of lung development and is responsible for the formation of the principle gas exchange units called alveoli. The lung mesenchyme, in particular the alveolar myofibroblasts, are drivers of alveolar development, however, few key regulators that govern the proper distribution and behavior of these cells in the distal lung during alveologenesis have been identified. While Hox5 triple mutants (Hox5 aabbcc) exhibit neonatal lethality, four-allele, compound mutant mice (Hox5 AabbCc) are born in Mendelian ratios and are phenotypically normal at birth. However, they exhibit defects in alveologenesis characterized by a BPD-like phenotype by early postnatal stages that becomes more pronounced at adult stages. Invasive pulmonary functional analyses demonstrate significant increases in total lung volume and compliance and a decrease in elastance in Hox5 compound mutants. SMA+ myofibroblasts in the distal lung are distributed abnormally during peak stages of alveologenesis and aggregate, resulting in the formation of a disrupted elastin network. Examination of other key components of the distal lung ECM, as well as other epithelial cells and lipofibroblasts reveal no differences in distribution. Collectively, these data indicate that Hox5 genes play a critical role in alveolar development by governing the proper cellular behavior of myofibroblasts during alveologenesis.


Funded by

a Ruth L. Kirschstein National Research Service Award(NSRA)

and Blood Institute(NHLBI)

Lung


Acknowledgment

This work was supported by a Ruth L. Kirschstein National Research Service Award (NSRA) training Grant 5 T32 HL 7749-20 to S.M.H. This research was also supported by MICHR PTSP UL1TR002240 to L.M.S and the National Heart, Lung, and Blood Institute (NHLBI) R01-HL119215 to D.M.W.


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Table S1 Primer sequences used

Figure S1 Hox5 AabbCc compound mutants have normal trachea at E18.5. Skeletal preparations of compound Hox5 AabbCc mutant mice reveal no phenotypic changes of the trachea (A) compared to the clear tracheal defects observed in Hox5 aaBBCC single mutants (B) at E18.5. Scale bar represents 1.0 mm.

Figure S2 qPCR of ECM components in distal airway of Hox5 AabbCc compound mutants at P7. qPCR measures no difference in the expression levels of several laminin mRNAs, Col4a1 and Col4a2, Col3a1, and fibronectin in Hox5 AabbCc mutants at P7. n=3 control and Hox5 AabbCc mutant animals were measured in qPCR analyses.

Supplemental movie1 (S1) 3D reconstruction of α-SMA myofibroblasts (red) in the distal lung region of control animals at P7.

Supplemental movie2 (S2) 3D reconstruction of α-SMA myofibroblasts (red) in the distal lung region of Hox5 AabbCc mutants animals at P7.

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    Hox5 four-allele compound mutants (Hox5 AabbCc) exhibit alveolar simplification during early postnatal and adult stages. Newborn Hox5 compound mutants are histologically indistinguishable from controls and have no measurable difference in alveolar chord length (A–B’; G). Scale bars in B, B’ represent 200 and 100 µm, respectively. By two weeks of age, Hox5 compound mutants exhibit enlarged, simplified alveoli compared to controls with a statistically significant ~33% increase in alveolar chord length (C–D’; G). Scale bars in C, C’ represent 200 and 100 µm, respectively. The most severe alveolar simplification phenotype of Hox5 AabbCc mutants occurs at adult stages, evidenced by the greatest increase in alveolar chord length (~100%) compared to controls (E–F’; G). Scale bars in F, F’ represent 400 and 100 µm, respectively. n=4 animals were measured for both control and Hox5 AabbCc groups for each stage depicted. P values and statistical significance (*) were determined by an unpaired Student’s t test.

  • Figure 2

    Hox5 AabbCc adults exhibit abnormal pulmonary function. Quasistatic and fast flow maneuvers were performed using the Buxco system to measure lung volume, compliance and elastance in tracheotomized mice. Increased lung volumes of Hox5 AabbCc mice are indicated by significant increases in inspiratory capacity (IC) (A), vital capacity (VC) (B), forced vital capacity (FVC) (C) and forced expiratory volume (FEV) (D). Increased lung compliance of Hox5 AabbCc mutants are determined by significant increases in chord compliance (Cchord) (E), compliance at zero pressure (Cp0) (F) and peak compliance (Cpk) (G). Hox5 AabbCc animals also exhibit a significant decrease in elastance at adult stages (H). n=4 control and Hox5 AabbCc animals were measured for PFT analyses. P values and statistical significance (*) were determined by an unpaired Student’s t test.

  • Figure 3

    Major ECM components of the distal airway are unaffected in Hox5 AabbCc mutants at P7. IHC analyses reveal no changes in the protein expression patterns of basement membrane components laminin (A, B) and Col4 (C, D) in Hox5 AabbCc mutants compared to controls. We also see no differences in the expression patterns of Col3 (E, F) or fibronectin (G, H) in the distal airway of Hox5 compound mutants at P7. Scale bars represent 50 µm.

  • Figure 4

    The elastin-based network is perturbed in Hox5 AabbCc mutants at P7. The elastin network forms tightly organized bundles that are localized around the alveolar openings and septal tips in control animals at P7 (A, A’). In contrast, the elastin network in Hox5 compound mutants is highly disorganized with entangled elastin fibers within the thickened lung parenchyma in the distal airway (B, B’). Scale bars in B, B’ represent 100 and 50 µm, respectively.

  • Figure 5

    Alveolar myofibroblasts are abnormally localized in the distal airway of Hox5 AabbCc mutants at P7. Control animals show a highly organized network of interconnected SMA+ alveolar myofibroblasts at P7 that are relatively evenly distributed around the alveolar openings and septal tips (A). In contrast, the network of SMA+ alveolar myofibroblasts is markedly more diffuse in Hox5 AabbCc mutants compared to controls with abnormal distribution and aggregation of these cells within the thickened lung parenchyma (A). Scale bars in (A) represent 200, 100 and 50 µm (left to right). SMA+ alveolar myofibroblasts continue to be tightly associated with elastin in Hox5 AabbCc mutants at P7, even in the distal lung regions that exhibit parenchymal thickening and myofibroblast clumping (B). Scale bar in (B) represents 50 µm. As observed in control animals, all SMA+ myofibroblasts co-express PdgfrαGFP in Hox5 AabbCc mutants and quantification reveals no difference in the total number of double positive cells at P7 (C). Scale bars in (C) represent 100 and 50 µm (left to right). n=3 control and Hox5 AabbCc mutant animals were used for quantification analyses.

  • Figure 6

    Additional cell types of the distal lung airway are unaffected in Hox5 AabbCc mutants at P7. IHC analyses reveal no differences in the distribution or morphology of T1α+ AECI cells (A, B) or Sftpc+ AECII cells (C, D) in Hox5 compound mutants. Adrp+ lipofibroblasts are normally distributed in the distal airway of Hox5 AabbCc mutants (E, F). Scale bars represent 50 µm. qPCR measures no differences in the expression levels of Elastin, T1α, Sftpc or Adrp in Hox5 compound mutants at P7. n=3 control and Hox5 AabbCc mutant animals were measured in qPCR analyses.

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